The oligopeptide transport system is essential for the development of natural competence in Streptococcus thermophilus strain LMD-9

Abstract

In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

FIG. 1.

Development of competence during growth of strain LMD-9 in CDM using pG⁺host9 plasmid as transformant DNA. The OD₆₀₀ (×, dashed line) was used to measure cell numbers and count of cells resistant to erythromycin (Ery-resistant cells) (⧫, plain line) was used to assess competence. Then, 1 μg of plasmid DNA was mixed with 100 μl of cells. The means of three independent experiments are presented, and error bars indicate standard deviations.

FIG. 2.

Development of competence during growth of strain LMD-9 in CDM using chromosomal DNA of strain TIL1192 as transformant DNA. The OD₆₀₀ (×, dashed line) was used to measure cell number and count of cells resistant to erythromycin (Ery-resistant cells) (⧫, plain line) was used to assess competence. Then, 1 μg of chromosomal DNA was mixed with 100 μl of cells. The means of four independent experiments are presented, and error bars indicate the standard deviations.

FIG. 3.

Relative expression levels of comX, recA, dpr, and comGA between S. thermophilus LMD-9 and strain TIL883 (LMD9 ΔamiCDE) or strain TIL1196 (LMD-9 comX::erm). Relative expression levels were computed by using the comparative critical threshold method (2^−ΔΔC_T) as described by Livak and Schmittgen (27). The data are expressed as means from three independent experiments and were significant according to an analysis of variance (P < 0.05).